Chitosan film with ternary metal oxides

ABSTRACT

A chitosan film including ternary metal oxides. In an embodiment, the ternary metal oxides can include tungsten oxide nanoparticles (WO3 NPs), magnesium oxide nanoparticles (MgO NPs), and a graphene oxide (GO) layer. In an embodiment the chitosan film is porous. The chitosan film including ternary metal oxides can be used as a wound dressing for the clinical management of bacterially infected wounds.

BACKGROUND 1. Field

The present disclosure relates to a chitosan membrane for wound healing,and in particular to a chitosan membrane with ternary metal oxides foruse in wound healing.

2. Description of the Related Art

Wound healing is a complex process where the skin or another organ ortissue repairs itself after injury. Once the protective barrier isbroken, an organism is susceptible to bacterial infections. Chronicbacterial infections in wounds pose a significant challenge tohealthcare, hindering healing and causing complications.

Chitin and chitosan, and their derivatives, are endowed with interestingchemical and biological properties. Chitosan is a deacetylatedderivative of chitin, a plentiful substance readily isolated from theshells of crustaceans such as crab, lobster, and shrimp. Antimicrobialproperties of chitosan open the door to a host of medical applications,such as antimicrobial coatings for adhesive strips or wound dressings.However, products having such properties remain under development andhave yet to be optimized.

Thus, a chitosan film including a ternary metal oxide solving theaforementioned problems are desired.

SUMMARY

The present subject matter relates to a chitosan (CS) composite filmincluding ternary metal oxides. In an embodiment, the ternary metaloxides can include tungsten oxide nanoparticles (WO₃ NPs), magnesiumoxide nanoparticles (MgO NPs), and a graphene oxide (GO)layer. In anembodiment the chitosan film is porous. The chitosan film includingternary metal oxides can be used as wound dressings for the clinicalmanagement of bacterially infected wounds.

In one embodiment, the present subject matter relates to a chitosancomposite film, comprising: a polymeric matrix including chitosan; and aplurality of ternary metal oxides comprising tungsten oxidenanoparticles (WO₃ NPs), magnesium oxide nanoparticles (MgO NPs), andgraphene oxide (GO) incorporated in or on the polymeric matrix.

In an embodiment, the chitosan composite film includes a polymericmatrix including chitosan, tungsten oxide nanoparticles (WO₃ NPs),magnesium oxide nanoparticles (MgO NPs), and a graphene oxide (GO) layeron the polymeric matrix. The graphene oxide thin layer can encouragecharge carriers that might enhance antibacterial performance.

According to an embodiment, the present subject matter relates to achitosan composite film includes a polymeric matrix including chitosan,tungsten oxide nanoparticles (WO₃ NPs) embedded within the polymericmatrix, magnesium oxide nanoparticles (MgO NPs) disposed on a surface ofthe polymeric matrix, and a graphene oxide (GO)layer on the polymericmatrix. The graphene oxide thin layer can encourage charge carriers thatmight enhance antibacterial performance.

According to an embodiment, the present subject matter relates to achitosan composite film, includes a polymeric matrix including chitosan;and a plurality of ternary metal oxides including tungsten oxidenanoparticles (WO₃ NPs) having an average diameter ranging from about 80nm to about 100 nm, magnesium oxide nanoparticles (MgO NPs) having anaverage diameter ranging from about 40 nm to about 50 nm, and a grapheneoxide (GO) layer on the polymeric matrix.

In a further embodiment, the present subject matter relates to a wounddressing material comprising a chitosan composite film as describedherein.

In one more embodiment, the present subject matter relates to a methodof healing a wound in a patient comprising applying to a patient in needthereof a chitosan composite film and/or a wound dressing material asdescribed herein at a site of the wound in the patient.

These and other features of the present subject matter will becomereadily apparent upon further review of the following specification.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows XRD patterns of pristine chitosan and WO₃, MgO, WO₃/MgO,and WO₃/MgO/GO nanocomposite powder materials loaded in a CS polymermatrix (CS, WO₃@CS, MgO@CS, WO₃/MgO@CS, and WO₃/MgO/GO@CS,respectively).

FIG. 2 shows FTIR spectra of the nanocomposites based on a CS polymermatrix including WO₃, MgO, WO₃/MgO, and WO₃/MgO/GO.

FIGS. 3A-3F show cross-sectional SEM images of (FIG. 3A) and (FIG. 3D)pure CS, (FIG. 3B) and (FIG. 3E) WO₃/MgO@CS, and (FIG. 3C) and (FIG. 3F)WO₃/MgO/GO@CS composites.

FIG. 4 shows an EDX graph of the WO₃/MgO/GO@CS composite.

FIGS. 5(A)-5(B) show (FIG. 5A) UV-Vis spectra of the synthesized CS andWO₃/MgO@CS, and (FIG. 5B) their energy band gap calculations from UV-Visspectra data.

FIGS. 6A-6F are images showing dependency of contact angle on thecompositional changes, with the values being 54.3°, 69.8°, 55.3°, and40.2° for (FIG. 6A) CS, (FIG. 6B) WO₃@CS, (FIG. 6C) MgO@CS, (FIG. 6D)WO₃/MgO@CS, and (FIG. 6E) WO₃/MgO/GO@CS, respectively, and (FIG. 6F) agraph showing the contact angle of pure CS membrane and CS membraneloaded with WO₃, MgO, WO₃/MgO, and WO₃/MgO/GO.

FIG. 7 is a graph showing cell viability of the WO₃/MgO/GO@CS compositeafter culturing for 3 days with normal lung cells in vitro.

FIGS. 8A and 8B are images showing the antibacterial activity for allscaffolds against (FIG. 8A) E. coli, and (FIG. 8B) S. aureus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions are provided for the purpose of understandingthe present subject matter and for construing the appended patentclaims.

Definitions

It should be understood that the drawings described above or below arefor illustration purposes only. The drawings are not necessarily toscale, with emphasis generally being placed upon illustrating theprinciples of the present teachings. The drawings are not intended tolimit the scope of the present teachings in any way.

Throughout the application, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present teachings can alsoconsist essentially of, or consist of, the recited components, and thatthe processes of the present teachings can also consist essentially of,or consist of, the recited process steps.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components. Further, it should be understood that elements and/orfeatures of a composition or a method described herein can be combinedin a variety of ways without departing from the spirit and scope of thepresent teachings, whether explicit or implicit herein.

The use of the terms “include,” “includes”, “including,” “have,” “has,”or “having” should be generally understood as open-ended andnon-limiting unless specifically stated otherwise.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise. In addition, where the use of theterm “about” is before a quantitative value, the present teachings alsoinclude the specific quantitative value itself, unless specificallystated otherwise. As used herein, the term “about” refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges,percentage ranges, or ratio ranges, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the described subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and such embodiments are alsoencompassed within the described subject matter, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use“comprising” language. However, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of”.

“Subject” as used herein refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, and petcompanion animals such as household pets and other domesticated animalssuch as, but not limited to, cattle, sheep, ferrets, swine, horses,poultry, rabbits, goats, dogs, cats and the like.

“Patient” as used herein refers to a subject in need of treatment of acondition, disorder, or disease, such as an acute or chronic airwaydisorder or disease.

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

In an embodiment, the present subject matter relates to a chitosan (CS)composite film including ternary metal oxides (also referred to hereinas “WO₃/MgO/GO@CS”). In an embodiment, the ternary metal oxides caninclude tungsten oxide nanoparticles (WO₃ NPs), magnesium oxidenanoparticles (MgO NPs), and a graphene oxide (GO) layer. In anotherembodiment, the tungsten oxide nanoparticles (WO₃ NPs), magnesium oxidenanoparticles (MgO NPs), and graphene oxide (GO) can be in or on apolymer matrix comprising the chitosan. The chitosan film includingternary metal oxides can be used as a wound dressing for the clinicalmanagement of bacterially infected wounds.

In one embodiment, the chitosan film including ternary metal oxides maybe incorporated into a wound dressing or otherwise used in medicalapplications. Such films may be produced by drying a stock solutionincluding chitosan and the ternary metal oxides.

According to an embodiment, the chitosan film including ternary metaloxides is porous. As described herein, addition of WO₃, MgO, and GO to achitosan film pr chitosan polymeric matrix can result in formation of aporous film with a relatively rough surface. In an embodiment, at leastsome of the nanoparticles can be disposed on a surface of the chitosanfilm, resulting in a relatively rough film surface. The chitosan filmalone can have pores with dimensions ranging from about 0.8 μm to about1.2 μm, on average. The MgO NPs can confine the flexibility of chitosanchains and make the film surface unsmooth with many minute granules andenlarged pores.

In an embodiment, the MgO NPs can be on a surface of the chitosanpolymeric matrix and have a diameter ranging from about 40 nm to about50 nm. In an embodiment, the WO₃ NPs can be embedded within the chitosanfilm. In an embodiment, the WO₃ NPs can have an average size rangingfrom about 80 nm to about 100 nm.

In an embodiment, the composite film can include a thin graphene oxide(GO) layer on a surface of the chitosan polymeric matrix. The grapheneoxide layer can encourage charge carriers that might enhanceantibacterial performance. In addition, incorporation of the thin GOlayer can result in a composite with a more homogeneous surface and aslight increase in roughness.

According to an embodiment, the film can be hydrophilic. Accordingly,when incorporated in a wound dressing material, the film can offer amoist environment at the wound surface, which would aid in adhesion ofthe wound dressing material over the wounded area.

In an embodiment, the present subject matter relates to a wound dressingor a wound dressing material comprising a chitosan composite film asdescribed herein.

In certain embodiments, the present wound dressings may comprise one ormore further excipients, carriers, or vehicles. Non-limiting examples ofsuitable excipients, carriers, or vehicles useful herein include liquidssuch as water, saline, glycerol, polyethyleneglycol, hyaluronic acid,ethanol, and the like. Suitable excipients for nonliquid formulationsare also known to those of skill in the art. A thorough discussion oftherapeutically acceptable excipients and salts useful herein isavailable in Remington's Pharmaceutical Sciences, 18th Edition. Easton,Pa., Mack Publishing Company, 1990, the entire contents of which areincorporated by reference herein.

The present wound dressings typically contain a therapeuticallyeffective dosage, e.g., a dosage sufficient to promote wound healing.

While human dosage levels have yet to be optimized for the present wounddressings, generally, a single wound dressing may contain from about0.01 to 10.0 mg/kg of body weight, for example about 0.1 to 5.0 mg/kg ofbody weight. The precise effective amount will vary from subject tosubject and will depend upon the species, age, the subject's size andhealth, the nature and extent of the condition being treated,recommendations of the treating physician, and the therapeutics orcombination of therapeutics selected for administration. The subject maybe administered as many wound dressings as is required to reduce and/oralleviate the wound.

The present wound dressings have valuable therapeutic properties, whichmake them commercially utilizable.

According to an embodiment, the film can be prepared by adding tungstenoxide (WO₃) nanoparticles, magnesium oxide (MgO) nanoparticles, andgraphene oxide (GO) to a solution including chitosan (CS) and stirringto provide a well-dispersed final mixture. In an embodiment, thesolution including chitosan can include deionized water and acetic acid.The final mixture can then be dried to provide the film. In certainembodiments, the final mixture can be washed before it is dried.

In an embodiment, the WO₃ NPs and MgO NPs can be prepared by anyconventional route. Similarly, the GO can be prepared using any suitablemethod known in the art, e.g., Hummer's method. In one embodiment, theGO can be prepared by combining graphite powder with a blend ofconcentrated H₂SO₄/H₃PO₄ under stirring to provide a mixture. Then,KMnO₄ can be added to the mixture with persistent stirring, followed byaddition of H₂O₂ to provide a final solution The final solution can bewashed with 30% HCl, then with distilled water, and finally withethanol. The washed solution can then be dried in a furnace to providegraphene oxide.

In a further embodiment, the present subject matter relates to a methodof healing a wound in a patient comprising applying to a patient in needthereof a chitosan composite film, a wound dressing, or a wound dressingmaterial as described herein at a site of the wound in the patient.

In certain embodiments in this regard, the wound dressing can stimulatecell proliferation at the site of the wound in the patient.

In another embodiment, application of the wound dressing can promotemetabolite circulation and vascularization.

In one embodiment of the present methods, the wound dressing can preventa microbial, bacterial, and/or fungal infection at the site of the woundin the patient. By way of non-limiting example, the present methods canprevent infection by E. coli and/or S. aureus at the site of the woundin the patient.

In another embodiment of the present methods, the wound dressing canpromote wound healing through a heightened ability to adhere to asurface of the wound.

In the above methods, the patient is preferably a mammal, morepreferably a human. Furthermore, the present wound dressing can be usedin combination therapy with one or more additional therapeutic agents.

The present teachings are illustrated by the following examples.

EXAMPLES Example 1 Preparation of Chitosan Film Including Ternary MetalOxides

First, WO₃ and MgO NPs were obtained. Graphene oxide was prepared byHummer's method, as follows. A graphite powder (3 g) was added to a 9:1blend of concentrated H₂SO₄/H₃PO₄ under stirring for 5 minutes. Then, 18g of KMnO₄ was added to the mixture with persistent stirring for 12 h.At that point, 3 mL of H₂O₂ was added and mixed for 1 h. The solutionwas washed with 30% HCl, then with distilled water, and finally withethanol, before it was dried in a furnace.

10 wt. % of a chitosan (CS) solution was prepared, and 5 samples of CSwere prepared as stock, each comprising 20 mL of deionized water/aceticacid (9/1 v/v). The first sample was pure CS without any additives. Thesecond sample included 0.25 g of WO₃ nanoparticles added to 10 mL of CSsolution. The third sample included MgO nanoparticles (0.25 g) added to10 mL of CS solution. The fourth sample included 0.125 g from WO₃ NPsand MgO NPs added to 10 mL of CS solution. Finally, the fifth sampleincluded 0.1 mg, 0.1 mg, and 0.05 mg of WO₃ and MgO NPs, and GO,respectively, added to 10 mL of CS solution. The additives were droppedinto each bottle and the solutions in each bottle were stirred using amagnetic stirrer for 1 hour to obtain well-dispersed solutions.

The solution was poured into Petri plates. An upside-down funnel wasused to cover each Petri plate. Overnight, the system was left to drynaturally. The samples were collected and kept in a desiccator untiltheir next usage after drying.

Example 2 Structural Analysis of Chitosan Film Including Ternary MetalOxides

The solution was poured into Petri plates. An upside-down funnel wasused to cover each Petri plate. Overnight, the system was left to drynaturally. The samples were collected and kept in a desiccator untiltheir next usage after drying.

FIG. 1 shows the XRD patterns of a pure CS membrane, WO₃@CS, MgO@CS,WO₃/MgO@CS, and WO₃/MgO/GO@CS composite membranes, respectively. Thesignificant peak of the CS membrane appeared at 21.9° whereas thesepeaks became frail in the XRD pattern of other composite membranes. Theintensive diffractions for the WO₃@CS composite appear at 23.56°,28.70°, 41.6°, and 55.8°. Accordingly, the consistent planes are (020),(111), (222), and (402), which confirm the establishment of themonoclinic phase of WO₃ nanoparticles that are compatible with the JCPDScard no. 89-4476. The XRD pattern of synthesized MgO@CS membrane iscomposed of 20 values at 31.34°, 36.78°, 42.73°, 45.08°, and 62.17°.These peaks disclose the occurrence of a high crystalline form of MgOnanoparticles (JCPDS Card No. 75-1525).

Moreover, the XRD for the last sample of WO₃/MgO/GO@CS confirmed theexistence of WO₃ and MgO in the composite in addition to acharacteristic peak at 10.8° and a fragile peak at 2θ=42 related to GOnanosheets due to (002) and C (100) planes respectively. It could beseen that the peaks of MgO@CS and WO₃@CS have different intensities whenthey are both with CS in the same film. This could be due to either thehigh dispersion of the nanoparticles in the film or the lowconcentration of the nanoparticles compared to the primary films and CSconcentration. These findings thus clearly demonstrate that thefunctionalization method was achieved efficiently through theincorporation of WO₃ and MgO NPs with GO nanosheets in the produced CSmembranes.

FT-IR spectrum of pure CS, WO₃/MgO@CS, and WO₃/MgO/GO@CS compositemembranes was studied in the region 400.0-4000.0 cm⁻¹ (FIG. 2 ). Itsdistinctive peaks can be found at 3413, 2936, 1644, 1314, and 1016 cm⁻¹,which were attributed to hydroxyl and amino stretching, C—H vibrations,acetylated amino groups, C—N bond stretching, and C—O—C bond stretching,respectively, identified Chitosan. Furthermore, in the WO₃/MgO@CS curve,the broad absorption peaks at less than 1000 cm⁻¹ specify the existenceof pure WO₃. The vibration band at 3413 cm⁻¹, which belongs to thehydroxyl groups of chitosan, has migrated to 3244 cm⁻¹ and 3283 cm⁻¹ byadding MgO and WO₃, respectively. The vibration modes of bond stretchingof O—W—O are ascribed to the peaks at 824 and 791 cm⁻¹. Additionally,the chitosan amine groups at 1644 cm⁻¹ have moved to 1550 cm⁻¹. Previousreports have studied similar changes in the vibrational bands of theamine groups of chitosan with various metal oxides. These shiftsproposed that chitosan's hydroxyl and amine groups might engage inhydrogen bonding interactions with MgO. By facilitating the effectivetransmission of stresses from the matrix to the filler under tensilestrain, this improved interfacial contact between MgO, WO₃, and chitosanenhances the mechanical characteristics of the resulting membrane. Dueto the very small concentration of GO in the latter composite, there isno clear characteristic peak associated with GO, most likely, the peakwas confused with the amino group peak for chitosan, but its presencewas previously confirmed by XRD analysis.

The morphology of the nanocomposite scaffold was examined by SEMinvestigation, and its results are presented in FIGS. 3A-3F. SEM is atechnique to study the surface morphology and topography of thematerial. According to SEM images, surface porosity, and roughness canbe studied. If the nanoparticles were embedded inside the film, thesurface roughness will be very low and the film surface will be smooth.On the other hand, the surface will be rough if some of thenanoparticles were on the surface of the film. FIGS. 3A-3C show thecross-section and FIGS. 3D-3F show the higher resolution images of pureCS (FIGS. 3A and 3D), MgO@CS (FIGS. 3B and 3E), and WO₃/MgO/GO@CS (FIGS.3C and 3F).

As shown in FIGS. 3A-3F, the inclusion of WO₃, MgO, and GOnanocomposites resulted in forming of all membranes with porousstructures and a relatively rough surface. The surface morphology ofpure CS only shows pores with dimensions of about (0.8 μm×1.2 μm) onaverage. The incorporation of MgO NPs into the membrane network confinesthe flexibility of chitosan chains and makes the composite membranesurface unsmooth with many minute granules and enhanced forming pores.MgO nanoparticles have diameters around 40-50 nm (FIG. 3B) and (FIG.3E). According to FIGS. 3C and 3F, the addition of WO₃ NPs tonanocomposite films disclosed distinct images (white dots) on the filmsurface, demonstrating the WO₃ NPs' excellent dispersion inside thepolymer matrices with an average size of 80-100 nm. As a result of theaddition of GO, a new graphene oxide thin layer formed in the sampleencouraged charge carriers that might enhance antibacterial performance.The surface of the composite became a more homogeneous surface with aslight increase in roughness (FIGS. 3C and 3F).

The EDX chart of the WO₃/MgO/GO@CS composite revealed the elementalcomposition of the sample (FIG. 4 ). As shown in FIG. 4 , distinct peaksare shown for all the constituent elements, including that of thecarbon, existing in GO and chitosan, the nitrogen existing in thechitosan, and both tungsten and magnesium. Correspondingly, the sharppeak of oxygen is assigned to the existence of WO₃ and MgOnanoparticles. The atomic percentages of all these elements areillustrated in Table 1.

TABLE 1 Elemental analysis of EDX for the composition of WO₃/MgO/GO@CSElement Weight % Atomic % C K 47.01 56.89 O K 44.63 40.55 MgK  3.66 2.19 WM  4.69  0.37

UV absorption of the chitosan nanocomposites is outlined in FIG. 5A.WO₃/MgO@CS composite has a much higher UV absorption than pure chitosanfilms. Referring to the pure CS spectra, neat chitosan films have a poorlimited UV absorption peak at 238 nm compared to that of WO₃/MgO/GO@CScomposite, which displays an absorption peak at 238 nm and a highintense absorption peak at 401 nm. These findings suggest that theUV-shielding properties of MgO nanoparticles caused the UV transmissionacross chitosan nanocomposite to be much lower than that through purechitosan films. Besides, WO₃ NPs emphatically affected the absorptionband of the WO₃/MgO/@CS composite and shifted towards the lowerwavelength region due to its small band gap (2.9 eV). These perceptionssupport the effective loading of WO₃ on CS and their interband chargetransition. To explore the impact of WO₃, MgO incorporation on theband-gap tuning of CS film Kubelka-Munk (KM) method was introduced. Theoptical absorption factor (a) can be defined as:

$\begin{matrix}{\alpha = {{F(R)} = \frac{\left( {1 - R} \right)^{2}}{2R}}} & (2)\end{matrix}$where F(R) is the Kubelka-Munk function of R, and R is the reflectioncoefficient (R=10−A). The gap-band energy of the compounds wascalculated from the plot between (ah v)2 vs. h v, as presented in FIG.5B. By modifying WO₃ and MgO NPs, a significant band gap narrowing wasobserved to be 3.69 ev. This can be related to the insertion of metallicnanoparticles within the created CS film, which causes a transition fromthe valence to the conduction band with less energy due to the creationof new interstitial energy levels.

Example 3 Contact Angle

Assessing a good dressing agent for tissue healing requires anunderstanding of how scaffolds interact with the surroundingenvironment. Furthermore, the compositional variation affects thewettability of the constructed scaffolds. The contact angle between thescaffold and the water droplets nearby indicates the scaffold'spotential to be coherent with the physiological milieu. As shown in FIG.6A, chitosan has a contact angle of about 54.31±2.46°, which indicatesthat its hydrophilicity is modest. The addition of WO₃ NPs caused acritical increment within the contact angle to be 69.84±0.51° (FIG. 6B),which decreased the hydrophilic properties of the scaffold. This can beattributed to the alteration within the surface morphology, which becamemore uniform and smoother with the addition of WO₃ NPs.

The same impact occurred by adding MgO NPs to the CS membrane, and thecontact angle slightly shifted to a higher value of 55.34±2.98° (FIG.6C). However, the incorporation of WO₃ and MgO NPs into the CS membrane(FIG. 6D) diminished the contact angle to almost 40.19±1.24°, whichinduces hydrophilicity and encourages their ability to be physicallycross-linked with the surroundings, thus controlling the ratio ofembedded nanoparticles through the scaffolds and playing a crucial rolein adjusting its hydrophilic properties. On the other hand, the additionof GO to the previous scaffold (FIG. 6E) showed an increase in thecontact angle to 48.74±3.39° which is still lower than the contact angleof pure CS. In conclusion, the created nanocomposite film may offer amoist environment at the wound surface, which would aid in the adhesionof the wound dressing material over the wounded area.

Example 4 Cell Viability

The percentage of viable cells reveals how the biological system reactsto the intended biomaterial. Therefore, the cell viability of human lungcells was evaluated using an optical analyzer (FIG. 7 ). Cell culturingis one of the direct tests to simulate the wounded area of the injuredperson. It is an effective test as it gives results not only close toreality but also, sufficient to know whether the scaffold is effectivein the biological medium or not. Cells are planted on the surface of thescaffold so that they can grow and multiply, since if they are plantedon the lower surface of the film, they will die.

The cell viability of WO₃/MgO/GO@CS nanocomposites was tested afterthree days. The fabricated composite achieved very high values of viablecells, 121.6%. Introducing WO₃ and MgO NPs increases the capability ofcell proliferation, with the highest IC₅₀ measured at 1654 μg/mL. Theviable cells proliferated and grew in a concentration of 2.44 μg/mL andreached a cell viability of 121.67%. Additionally, rough surfaces andporosity caused by NPs incorporation are vital for adhesion and feedingcells with blood. This demonstrates a potential of the tested compositein cell viability. Also, addition of GO to the CS membrane improved thesurface roughness, hydrophilicity, and surface area of CS scaffoldswithout destroying the structure of the membrane, which plays animportant role in enhancing the cell proliferation ratio of thecomposite. This result shows the incredible biocompatibility of thefabricated composite which is recommended for tissue engineeringapplications. The scaffolds have been cultured for 3 days as it isreal-time for the healing process. The cell viability trend showed anincrease in viable cells by increasing culturing days.

Example 5 Anti-Bacterial Activity

Antibacterial activity is an essential property for accelerating thewound healing process. Accordingly, it was necessary to measure theability of scaffolds to kill bacteria. For this purpose, theantibacterial activity of the scaffold was tested against 2 types ofbacteria Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus). According to FIGS. 8A-8B, the pure CS film showed antibacterialactivity against E. coli and S. aureus with an inhibition zone of11.5±0.5 mm and 12.5±0.5 mm, respectively.

On the other hand, addition of MgO to CS enhanced the antibacterialactivity of the scaffold against E. coli as the inhibition zoneincreased to 13±1 mm while it decreased against S. aureus reaching11.5±0.5 mm. Further, the addition of WO₃ showed zones of inhibitionequal to 8.5±0.5 mm and 10.5±0.5 mm against both E. coli and S. aureus.Additionally, the combination of both MgO and WO₃ in CS filmdemonstrated stability in the antibacterial activity of CS against E.coli 11.5±0.5 mm and furthermore, an increase in the antibacterialbehavior against S. aureus with a zone of inhibition equal to 13.5±0.5mm.

Finally, GO was added to the previous film with a very lowconcentration. The antibacterial activity of the scaffold against E.coli plateaued, while it exhibited a decrease in the behavior against S.aureus reaching 8.5±0.5 mm. In conclusion, it could be seen that all thescaffolds have antibacterial activity against both types of bacteria.The highest and best antibacterial activity was the scaffold of thechitosan composite film (referred to as MgO/WO₃@CS).

It is to be understood that the chitosan composite film is not limitedto the specific embodiments described above but encompasses any and allembodiments within the scope of the generic language of the followingclaims enabled by the embodiments described herein, or otherwise shownin the drawings or described above in terms sufficient to enable one ofordinary skill in the art to make and use the claimed subject matter.

We claim:
 1. A chitosan composite film, comprising: a polymeric matrixincluding chitosan; and a plurality of ternary metal oxides comprisingtungsten oxide nanoparticles (WO₃ NPs), magnesium oxide nanoparticles(MgO NPs), and graphene oxide (GO) incorporated in or on the polymericmatrix.
 2. The chitosan composite film of claim 1, wherein the chitosancomposite film is porous.
 3. The chitosan composite film of claim 1,wherein at least some of the MgO NPs are disposed on a surface of thepolymeric matrix.
 4. The chitosan composite film of claim 1, wherein theWO₃ NPs are embedded within the polymeric matrix.
 5. The chitosancomposite film of claim 1, wherein the MgO nanoparticles have an averagediameter ranging from about 40 nm to about 50 nm.
 6. The chitosancomposite film of claim 1, wherein the WO₃ NPs have an average diameterranging from about 80 nm to about 100 nm.